The present invention relates to identification and characterisation of Islet-Brain 1 (IB1), a transcriptional activator that is involved in the control of the GLUT2 and insulin genes by interacting with homologous cis-regulatory elements of the GLUT2 and insulin promoters, and to materials and methods deriving from this work. In particular, the present invention relates to the uses of IB1 nucleic acid, IB1 polypeptides, IB1 antagonists and antibodies in the diagnosis, and prophylactic and therapeutic treatment of conditions such as diabetes, neurological diseases such as dementia and/or parkinsonism, cancer and in the promotion or inhibition of apoptosis.
The GLUT2 facilitated glucose transporter isoform is a membrane protein present in the pancreatic xcex2-insulin-secreting cells, the basolateral membrane of intestinal and kidney absorptive cells, in hepatocytes and in a subset of neurons (21,31,44). In these cells, GLUT2 catalyzes the transepithelial transport of glucose. In pancreatic islets, GLUT2 allows a rapid equilibration of glucose between the extracellular space and the interior of the cells and it may play a crucial role in the glucose signalling mechanism leading to insulin secretion (43). However, the relative importance of GLUT2 in the sensing of the xcex2-pancreatic cells to glucose remains debated. In human xcex2-cells, the level of expression of GLUT2 is low and the intracellular glucokinase activity seems to be the rate-limiting step in the glycolytic pathway (5,11). On the other hand, insulinoma cells that had lost their normal glucose responsiveness have low GLUT2 content, but some glucose sensitivity may be recovered after reintroducing GLUT2 expression through stable transfection of these cells (10,16). Furthermore, transgenic mice that express GLUT2 antisense RNAs driven by the insulin promoter led to an 80% reduction in GLUT2 which was paralleled by a decreased glucose-induced insulin secretory response and by the onset of diabetes (48). These observations are critical since several experimental models of diabetes have shown that GLUT2 expression is dramatically reduced specifically in the pancreatic xcex2-cells, and that this mechanism could participate to the onset of the disease (18,29,30,32,45-47). Therefore, while GLUT2 levels are unchanged or even upregulated in several tissues such as the liver and the intestine during the hyperglycemic conditions observed in diabetes, the same gene undergoes a drastic dysregulation only in the pancreatic islets.
A fragment of the murine GLUT2 promoter has been cloned and shown to be glucose-responsive when transfected into differentiated insulin-producing cells or into hepatocytes (35,36,52). Important cis-regulatory sequences were identified within this promoter region including a functionally responsive PDX-1 element, a cyclic AMP responsive element, and three cis-elements termed GTI, GTII and GTIII (3,36,53). The presence of GTI, II and III are both sufficient and necessary to confer pancreatic-specific expression to a reporter gene in vitro or in vivo, using a transgenic mice approach (3,51). GTI and GTIII have been previously shown to bind distinct, but ubiquitously expressed trans-acting factors.
The present invention is based on successful expression cloning of a transcription factor that binds to the GTII element of the GLUT2 and insulin genes from a differentiated insulin-secreting cDNA library. In part, the success of this exercise was based on the inventors"" realisation of the importance of GTII and the library they constructed to find the IB1 gene. The IB1 polypeptides described herein are relatively large, and cloning them was achieved by the construction by the inventors of a high quality cDNA library for expression cloning.
This factor is abundantly expressed in the pancreatic islets and in the brain and has been named IB-1 for Islet-Brain 1. Both human and rat IB1 genes and polypeptides have been obtained. The rat IB1 cDNA (SEQ ID NO: 1) encodes a 714 amino acid protein (SEQ ID NO: 2) and the human IB1 cDNA (SEQ ID NO: 3) a 711 amino acid protein (SEQ ID NO: 4). The cDNAs encoding the rat and human IB1 polypeptides have a 94% sequence identity and the polypeptides have a 97% amino acid sequence identity (see the alignment of the sequences in FIG. 1D), and have a proline-rich region and a putative basic helix-loop-helix domain (bHLH). The IB1 gene is highly expressed in the pancreatic islets and in the brain and to a much lesser extent in the heart and the kidney. In the Langerhans islets and in xcex2-cell lines, these transcripts are translated into immunodetectable cytoplasmic and nuclear protein. When tested in vitro, IB1 bound specifically to the GTII cis element of the GLUT2 gene and to an homologous regulatory sequence of the insulin promoter termed RIPE3. This rat insulin promoter element 3 (RIPE3) is an important enhancer sequence sufficient to confer xcex2-cell specific expression to the insulin gene. Functionally, IB1 transactivated the proximal region of the GLUT2 promoter linked to a luciferase reporter gene and was also a potent activator of the insulin gene. This effect is mediated through the RIPE3 sequence as demonstrated by the observation that multiple copies of this enhancer sequence cloned 5xe2x80x2 of an heterologous promoter was transactivated by an expression vector encoding IB1 in transient transfection studies. IB1 appears to function only in insulin-secreting cells as no transactivation was observed in non-pancreatic or in glucagon-producing cell lines. These data demonstrate the presence of a novel transcriptional activator abundantly expressed in the endocrine pancreas and which participates to the proper xcex2-cell specific control of the GLUT2 and the insulin genes through homologous sequences present in both promoters.
The nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2) of rat IB1 are shown in FIG. 1A. The human IB1 cDNA (SEQ ID NO: 3) is shown in FIG. 1E, with the translated amino acid sequence (SEQ ID NO: 4) shown in FIG. 1F. The human IB1 gene is located on chromosome 11 at 11p11.12 on the LDB cytogenic map. The IB1 gene is adjacent to markers D11S134 and D11S3979.
The human cDNA was constructed as RNA using tissue obtained from a surgically removed human insulinoma. Poly A+ RNA was extracted and a cDNA library constructed and subsequently screened with a radiolabelled rat IB1 cDNA probe. This allowed the inventors to isolate the human cDNA encoding IB1. This cDNA was then used as a probe to clone the human IB1 gene from a bacterial artificial chromosome (BAC). Several clones were obtained and part of them sequenced. The above protocol was then used to complete the sequencing of the human IB1 nucleic acid shown in FIG. 1E (SEQ ID NO: 3).
The human IB1 gene is multiexonic and is located in chromosome 11p11.12. The chromosomal mapping was obtained by FISH experiments and PCR of hybrid cells (hamster-human) using as a probe the multiexonic IB1 gene. IB1 is expressed in the brain of rat, mouse and human species and in tissues with a high degree of similarity such as the endocrine pancreas (many neuronal features are present in the insulin xcex2-cells).
In a first aspect, the present invention provides a substance which is an isolated polypeptide comprising a polypeptide having the amino acid sequence set out in FIG. 1A (SEQ ID NO: 2) or FIG. 1F (SEQ ID NO: 4).
In a further aspect, the present invention provides a substance which is an isolated polypeptide having greater than 80% amino acid sequence identity with the amino acid sequence set out in FIG. 1A (SEQ ID NO: 2) or 1F (SEQ ID NO: 4).
In a further aspect, the present invention provides a substance which is a polypeptide which is a mutant, variant, derivative or allele of any one of the above polypeptides.
In a further aspect, the present invention provides a substance which is a fragment of a polypeptide having the amino acid sequence set out in FIG. 1A or 1F which exhibits a biological property of full length IB1 protein. In one embodiment, the fragment includes the domain from amino acids 566-612 (SEQ ID NO: 2) of the sequence shown in FIG. 1A or the domain from amino acids 563-609 (SEQ ID NO: 4) of the sequence shown in FIG. 1F, or an active portion of that domain. In an alternative embodiment, the present invention provides a polypeptide which is a protein interaction domain having the sequence shown in FIG. 1A from amino acids 566-612 (SEQ ID NO: 2) and in FIG. 1E from amino acids 563-609 (SEQ ID NO: 4). As this domain is believed to be responsible for some of the interactions between IB1 and other polypeptides, it can be used in methods of screening for binding partners, e.g. peptides which could act as inhibitors of IB1.
In a further aspect, the present invention provides isolated nucleic acid molecules encoding any one of the above polypeptides. Examples of such nucleic acid sequences are the nucleic acid sequences set out in FIGS. 1A (SEQ ID NO: 1) and 1E (SEQ ID NO: 3). The present invention also include nucleic molecules having greater than a 90% sequence homology with the nucleic acid sequence of FIG. 1A or 1E.
In further aspects, the present invention provides an expression vector comprising the above IB1 nucleic acid operably linked to control sequences to direct its expression, and host cells transformed with the vectors. The present invention also includes a method of producing IB1 polypeptides comprising culturing the host cells and isolating the IB1 polypeptide thus produced.
In a further aspect, the present invention provides an expression vector comprising IB1 nucleic acid for use in methods of gene therapy, e.g. in the treatment of patients unable to produce sufficient IB1 or to engineer cell lines capable of producing IB1.
In a further aspect, the present invention provides a cell line for transplantation into a patient, the cell line being transformed with nucleic acid encoding an IB1 protein, and being capable of producing IB1 polypeptide. The expression of IB1 in a transformed cell line can affect endogenous genes such as the insulin or GLUT2 genes. In one embodiment, the cell lines can be encapsulated, e.g. in a biocompatible polymer so that the IB1 produced by the cells line can be secreted into the patient, while preventing rejection by the immune system of the host. Methods for encapsulating cells in biocompatible polymers are described in WO93/16687 and WO96/31199.
In a further aspect, the present invention provides a pharmaceutical composition comprising an IB1 nucleic acid molecule.
In a further aspect, the present invention provides a pharmaceutical composition comprising one or more IB1 polypeptides as defined above.
In further aspects, the present invention provides the above IB1 polypeptides and nucleic acid molecules for use in methods of medical treatment. The present invention further provides the use of the IB1 polypeptides in the preparation of medicament for activating the GLUT2 or insulin promoters leading to the production of GLUT2 or insulin. Preferably, the activation takes place in a cell specific manner, e.g. in xcex2-cells. This could be used in the treatment of conditions treatable using insulin or GLUT2, such as diabetes. Since IB1 is also present in muscle tissue such as the heart it may function by modulating insulin sensitivity and ameliorating abnormal glucose disposal in diabetic patients.
IB1 could also be used as an agent which maintains a state of differentiation within in a cell, i.e. acts as an anti-apoptotic agent. Thus, IB1 can be used as an anti-neoplastic agent, e.g. as a drug to control or treat some cancers. As an example, insulinomas are human tumours which undergo dedifferentiation and divide. Thus, IB1 antagonists could be used to attack tumour cells, while IB1 could be used to protect surrounding healthy tissue from the effects of treating tumour cells, using IB1 antagonists and/or conventional radiotherapy or chemotherapy. Thus, IB1 could act as a differentiation agent to treat these cells. A further application of IB1 antagonists is in the treatment of brain tumours, such as glioblastomas which are typically untreatable using conventional medicine.
IB1 is similar to JIP-1, a cytoplasmic protein identified by Dickens et al,. Science, 277:693-696, 1997. However, IB1 differs from JIP-1 by the insertion of 47 amino acids in the carboxy terminal portion of the protein, and has a 97% amino acid homology over the remaining sequence of rat IB1 shown in FIG. 1A (i.e. excluding the insert not present in JIP-1). As overexpression of JIP-1 in neuronal cells inhibits apoptosis of the cells, IB1 could be used to suppress apoptosis in cells, e.g. stress-induced apoptosis induced in neurons. This stress activation can be caused by ultraviolet (uv) radiation, anoxia, hypoglycemia, cytokines such as IL-6, or by trauma.
This in turn supports the present inventors"" earlier suggestion to the use of IB1 to prevent cell death that occurs in diseases such as dementia, neurodegenerative diseases, ischemia of the heart, myocardial infarction (IB1 is present in heart), and post trauma, e.g. in sections of the spine in paraplegia and in neuronal trauma.
In a further aspect, the present invention provides the use of the IB1 polypeptides in screening candidate compounds for IB1 biological activity, e.g. to find peptidyl or non-peptidyl mimetics of the IB1 polypeptides to develop as lead compounds in pharmaceutical research.
In a further aspect, the present invention provides antibodies capable of specifically binding to the above IB1 polypeptides. These antibodies can be used in assays to detect and quantify the presence of IB1 polypeptide, in methods of purifying IB1 polypeptides, and in pharmaceutical compositions, e.g. to neutralize IB1 in conditions in which its overexpression has deleterious effect, or to inhibit the anti-apoptotic effect of IB1 in diseased tissue such as tumours. Accordingly, antagonists such as antibodies which block the expression of IB1 or neutralize IB1 in the tissues in which it is overexpressed can be used for treating such conditions and are included within the present invention. Polyclonal antibodies to the N-terminal portion (residues 1-280 (SEQ ID NO: 2)) of the IB1 protein of FIG. 1A are exemplified below.
In a further aspect, the present invention method for determining the presence of IB1 nucleic acid and/or mutations within an IB1 nucleic acid sequence in a test sample comprising detecting the hybridization of test sample nucleic acid to a nucleic acid probe based on the IB1 nucleic acid sequences set out in FIG. 1A or 1E.
In a further aspect, the present invention provides the use of IB1 nucleic acid as defined above in the design of antisense oligonucleotides to restrict IB1 expression in a population of cells, i.e. phosphorothiolated or chloresterol linked oligonucleotides which can facilitate internalization and stabilization of the oligonucleotides.
In a further aspect, the present invention provides a method of amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase reaction with nucleic acid encoding an IB1 polypeptide as defined above. The present invention also provides the use of the above nucleic acid in the search for mutations in the IB1 genes, e.g. using techniques such as single stranded conformation polymorphism (SSCP).
These and other aspects of the present invention are described in more detail below.